HIGHLY CONCENTRATED FORMULATIONS OF SOLUBLE Fc RECEPTORS

ABSTRACT

The present invention relates to novel formulations of soluble Fc receptors and especially to formulations containing high concentrations of soluble FcRIIB receptor. The invention further relates to the use of such formulations as pharmaceutical compounds for the treatment of autoimmune diseases, infections and other conditions where the immune system is involved.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/787,097, filed Oct. 26, 2015, which is a 371 of PCT/EP2014/001029,filed Apr. 16, 2014, which claims the benefit of EP Patent ApplicationNo. 13002211.4, filed Apr. 26, 2013. The entire contents of each ofthese applications are incorporated by reference herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the determination of denaturing pH range. 0.5 mg/mL husFc

RIIB in 20 mM histidine, 150 mM NaCl (o) and blank buffer (+) wereincubated at the respective pH for 3 h at room temperature. An increasein Sypro Orange fluorescence indicated the presence of denatured husFc

RIIB. husFc

RIIB did not unfold from pH 5.2 to at least pH 11.

FIGS. 2A and 2B depicts the crystallization of husFc

RIIB. husFc

RIIB crystallization in 10 mM histidine pH 6.7 (FIG. 2A) or 10 mMcitrate pH 5.5 (FIG. 2B) as a function of NaCl and sugar (2:1sucrose:mannitol) concentration. husFc

RIIB crystallizes more readily and the crystal yield is much higher inthe presence of 10 mM histidine, 10 mM NaCl pH 6.7 compared to 10 mMcitrate, 10 mM NaCl pH 5.5.

FIG. 3 depicts husFc

RIIB crystallization performed in 10 mM histidine, 10 mM NaCl as afunction of pH. husFc

RIIB in 10 mM histidine, 10 mM NaCl pH 5.5 was concentrated to 140 mg/mLby ultrafiltration and diluted to 40 mg/mL with appropriate stocksolutions. The crystal yield was determined by measuring the husFc

RIIB concentration in the supernatant after 3d at 2.8° C. Each solutioncontained 0.01% polysorbate 20.

FIG. 4A to 4D depicts melting temperature Tm of citrate buffered husFc

RIIB formulations measured by Differential Scanning Fluorimetry. Use of0.5 mg/mL husFc

RIIB in 10 mM citrate, 4.5% sugar (2:1 (w/w) sucrose:mannitol), 75 mMNaCl at the indicated pH (FIG. 4A and FIG. 4C), or 0.5 mg/mL husFc

RIIB in 10 mM citrate pH 7.0 supplemented with the indicated amount ofsugar (2:1 (w/w) sucrose:mannitol) and salt (FIG. 4B and FIG. 4D). husFc

RIIB formulations were heated in a 96 well microtiter plate at 1° C./minin the presence of Sypro Orange and the fluorescence emission at 610 nmwas recorded. The fluorescence vs. temperature plots (FIG. 4A and FIG.4B) and their first derivatives (FIG. 4C and FIG. 4D) are shown. Thefirst maximum in the dF/dT plots was defined as husFc

RIIB melting temperature.

FIG. 5 depicts the change of husFc

RIIB melting temperature as a function of pH, sugar and saltconcentration in 10 mM citrate. The average and standard deviation fromthree independent wells are shown.

FIG. 6A to 6C depicts turbidity of citrate buffered husFc

RIIB formulations. Optical density at 360 nm was measured as a functionof husFc

RIIB concentration and sucrose concentration after 1 h (FIG. 6A), 12h(FIG. 6B) and 7d (FIG. 6C). All formulations contained 10 mM citrate pH7.0, 25 mM NaCl. The optical density of a buffer control was subtracted.At 40% sucrose, the highest concentrated formulation contained only 60mg/mL husFc

RIIB and not 80 mg/mL.

FIG. 7A to 7B depicts accelerated stability of citrate buffered husFc

RIIB formulations at 40° C. The increase in optical density at 360 nmwas measured at 10 mg/mL husFc

RIIB in 10 mM citrate, 150 mM NaCl pH 7.0 supplemented with 10%/292 mMsucrose (FIG. 7A, A), 10%/292 mM trehalose (FIG. 7A, €), 5%/274 mMmannitol (FIG. 7A, ◯), 30%/876 mM sucrose (FIG. 7B, A), 30%/876 mMtrehalose (FIG. 7B, €), 15%/822 mM mannitol (FIG. 7B, ◯). The buffercontrol supplemented with 20% sucrose is also shown (⋅).

FIGS. 8A and 8B depicts determination of free polysorbate 20 in thepresence of husFc

RIIB. husFc

RIIB in 10 mM citrate, 25 mM NaCl, 3% sucrose, 1.5% mannitol, 0.005%polysorbate 20, pH 6.7 was incubated for 1 h at 25° C. (native husFc

RIIB) and 60° C. (denatured husFc

RIIB). After husFc

RIIB was removed by cation exchange chromatography (CEX), the amount ofpolysorbate 20 was measured (FIG. 8A). The blank represents bufferwithout added husFc

RIIB or detergent. The system suitability tests (SST) represent bufferwith 0.005% polysorbate 20, SST2 was CEX treated and SST1 not. Themeasured polysorbate content at 0.08-0.72 mg/mL husFc

RIIB was extrapolated to husFc

RIIB concentrations above 10 mg/mL by linear regression (R2>0.998) ofthe polysorbate concentration versus the logarithmic husFc

RIIB concentration (FIG. 8B).

FIG. 9 depicts solution viscosity of husFc

RIIB in 10 mM citrate, 25 mM NaCl, pH 7.0 was measured at 20° C. (◯) wasfitted to an exponential growth function (−, R2>0.992) and found thatthe solution viscosity of the formulation is low enough to run economicTFF processes up to at least 210 mg/mL.

FIG. 10 depicts particulate load of selected formulations as determinedby fluorescence microscopy. All formulations contained 5 mM citrate pH6.7 and the indicated amount of salt, sugar and detergent. Theformulations were lyophilized and reconstituted to the indicated husFc

RIIB content.

FIG. 11 shows Table 2; Histidine based solubility screen at 70 mg/mLhusFcγRIIB and 2-8° C. The visual appearance of each formulation wasassessed by light microscopy and ranked according to an arbitrary scale(0=no crystals; 1=some crystals, hardly visible; 2=some crystals clearlyvisible; 3=more than 30 crystals per well clearly visible; 4=layer ofmany crystals (well not fully covered); 5=layer of many crystals (wellcompletely covered).

FIG. 12 shows Table 3: Histidine based solubility screen at 100 mg/mLhusFc_(y)RllB and 2-8° C. The visual appearance of each formulation wasassessed by light microscopy and ranked according to an arbitrary scale(0=no crystals; 1=some crystals, hardly visible; 2=some crystals clearlyvisible; 3=more than 30 crystals per well clearly visible; 4=layer ofmany crystals (well not fully covered); 5=layer of many crystals (wellcompletely covered). FIG. 13 shows Table 4: Histidine based solubilityscreen at 120 mg/mL/150 mg/mL husFcγRIIB and 2-8° C. The visualappearance of each formulation was assessed by light microscopy andranked according to an arbitrary scale (0=no crystals; 1=some crystals,hardly visible; 2=some crystals clearly visible; 3=more than 30 crystalsper well clearly visible; 4=layer of many crystals (well not fullycovered); 5=layer of many crystals (well completely covered).

FIG. 14 shows Table 5: Citrate based solubility screen at 70 mg/mLhusFcγRIIB and 2-8° C. The visual appearance of each formulation wasassessed by light microscopy and ranked according to an arbitrary scale(0=no crystals; 1=some crystals, hardly visible; 2=some crystals clearlyvisible; 3=more than 30 crystals per well clearly visible; 4=layer ofmany crystals (well not fully covered); 5=layer of many crystals (wellcompletely covered).

FIG. 15 shows Table 6: Citrate based solubility screen at 100 mg/mLhusFcγRIIB and 2-8° C. The visual appearance of each formulation wasassessed by light microscopy and ranked according to an arbitrary scale(0=no crystals; 1=some crystals, hardly visible; 2=some crystals clearlyvisible; 3=more than 30 crystals per well clearly visible; 4=layer ofmany crystals (well not fully covered); 5=layer of many crystals (wellcompletely covered).

FIG. 16 shows Table 7: Citrate based solubility screen at 120 mg/mL/150mg/mL husFcγRIIB and 2-8° C. The visual appearance of each formulationwas assessed by light microscopy and ranked according to an arbitraryscale (0=no crystals; 1=some crystals, hardly visible; 2=some crystalsclearly visible; 3=more than 30 crystals per well clearly visible;4=layer of many crystals (well not fully covered); 5=layer of manycrystals (well completely covered).

DESCRIPTION

The present invention relates to novel formulations of soluble Fcreceptors and especially to formulations containing high concentrationsof a soluble FcγRIIB receptor. The invention further relates to the useof such formulations as pharmaceutical compositions for the treatment ofautoimmune diseases, infections, tumors and other conditions where theimmune system is involved.

Human soluble FcγRIIB is a promising candidate substance for treatmentof Idiopathic Thrombocytopenic Purpura, Systemic Lupus Erythematosus andother autoimmune diseases. It is one of a plurality of soluble antibodyreceptors which have been developed over the past 10 to 15 years.

WO 00/32767 describes soluble Fc receptors (sFcRs) which are composed ofonly the extracellular part of the receptor and are not glycosylated.Due to the absence of the transmembrane domain and the signal peptide,these proteins are present in a soluble form and not bound to cells asis normally the case for Fc receptors (FcRs). Furthermore the sFcRsdescribed in WO 00/32767 can be produced recombinantly and have beensuggested for the treatment of autoimmune diseases due to their abilityto bind the Fc part of antibodies without interfering with othercomponents of the immune system. WO 00/32767 additionally describes thecrystal structure of certain sFcRs and the possibility of developingsubstances that inhibit the interaction of IgG with sFcRs with the aidof these crystal structures. The elucidation of the crystal structureenables finding such inhibitors by e.g. screening the databases usingavailable computer programs or by computer-aided drug design.

The invention which was defined in WO 03/043648 further developed thefindings of WO 00/32767 and provides treatment methods especially fordiseases like multiple sclerosis (MS), systemic lupus erythematosus(SLE), and rheumatoid arthritis (RA) and also for diseases with anelevated level of natural killer (NK) cells. Even if said receptors wereproduced recombinantly in prokaryotes and therefore were unglycosylated,the inventors of WO 03/043648 surprisingly found that although theunglycosylated proteins were expected to be poorly soluble, thereceptors could be purified with relatively high concentrations of up to50 mg/ml of sFcγR in a soluble form.

WO 00/32767, WO 03/043648 and other publications imply an important rolefor FcRs in defense reactions of the immune system. When pathogens haveentered the blood circulation they are bound by immunoglobulins, alsoknown as antibodies. Since the immune response to a pathogen ispolyclonal, a multitude of antibodies are produced and bind to apathogen, leading to the formation of an immune-complex (IC). ICs aresubsequently phagocytised by specialized effector cells (e.g. phagocytesor macrophages) of the immune system and thus removed from thecirculation. The phagocytosis is mediated by the binding of the Fc-partof the antibodies, which, together with the pathogen, form the ICs, toFcRs on the aforementioned effector cells. Other effector cells of theimmune system, such as natural killer cells, eosinophils and mast cellsalso carry FcRs on their surface which upon binding of ICs releasestored mediators such as growth factors or toxins that support theimmune response.

The FcRs of these effector cells also function as signal-transducingmolecules that specifically bind immunoglobulins of various isotypesduring the humoral immune response. In addition, FcRs expressed onnatural killer cells play a fundamental role in the destruction ofantibody-coated target cells (“antibody-dependent cell-mediatedcytotoxicity”, ADCC).

However, in addition to the positive effects of FcRs in the defenseagainst pathogens, overshooting reactions caused by the presence ofauto-antibodies in patients may also occur which result in an undesiredstimulation of the immune system which manifests itself especially asinflammatory or autoimmune diseases. Such immune reactions directedagainst the body's own substances remain a major medical problem andalthough there are approaches for treating them, these approaches arenot equally effective in every patient.

All members of the FcγR-family, i.e. FcRs which are specific forantibodies of the IgG type, are integral membrane glycoproteins,possessing extracellular domains related to a C2-set ofimmunoglobulin-related domains, having a single membrane spanning domainand an intracytoplasmic domain of variable length. There are three knownFcγ receptor forms, designated FcγRI (CD64), FcγRII (CD32), and FcγRIII(CD16). This invention in preferred embodiments specifically focuses onFcγRII (CD32).

FcγRII proteins are 40 KDa integral membrane glycoproteins which onlybind the complexed IgG in the ICs. These receptors are the most widelyexpressed FcγRs, present on all hematopoietic cells, includingmonocytes, macrophages, B cells, NK cells, neutrophils, mast cells, andplatelets. There are three human FcγRII genes (FcγRII-a, FcγRII-b,FcγRII-c), all of which bind IgG in aggregates or immune complexes.

Inflammation is a process by which the body's white blood cells react toinfection by foreign substances, such as bacteria and viruses. It isusually characterized by pain, swelling, warmth and redness of theaffected tissue. Effector substances known as cytokines andprostaglandins control this process, and are released in an ordered andself-limiting cascade into the blood or affected tissues. The release ofsuch effector substances increases the blood flow to the area of injuryor infection. Some of the effector substances cause a leak of fluid intothe tissues, resulting in swelling. This protective process maystimulate nerves and cause pain. These changes, when occurring for alimited period in the relevant area, work to the benefit of the body.

In autoimmune diseases the patient's immune system has lost the abilityto discriminate between body-own (“self”) and foreign proteins. Inconsequence, antibodies are generated that recognize “self”-proteins andform immune complexes which continuously activate the immune systembecause the “self”-protein is permanently produced and recognized asforeign. This chronic condition can persist for years leading in the endto severe organ damage and possibly to the death of the patient. Thereare many different autoimmune disorders which affect the body in variousways. For example, the brain is affected in individuals with multiplesclerosis, the gut is affected in individuals having Crohn's disease,and the synovium, bone and cartilage of various joints are affected inindividuals suffering from rheumatoid arthritis. As autoimmune disordersprogress, destruction of one or more types of body tissues, abnormalgrowth of an organ, or changes in organ function may result. Theautoimmune disorder may affect a single organ or tissue type or mayaffect multiple organs and tissues. Organs and tissues commonly affectedby autoimmune disorders include red blood cells, blood vessels,connective tissues, endocrine glands (e.g. the thyroid or pancreas),muscles, joints, and the skin.

Examples of inflammatory and/or autoimmune disorders include, but arenot limited to, primary immune thrombocytopenia (ITP), systemic lupuserythematosus (SLE), rheumatoid arthritis (RA), autoimmune haemolyticanaemia (AIHA), diabetes, Pemphigus vulgaris, Hashimoto's thyroiditis,autoimmune inner ear disease myasthenia gravis, pernicious anemia,Addison's disease, dermatomyositis, Sjogren's syndrome, dermatomyositis,multiple sclerosis, Reiter's syndrome, Graves disease, autoimmunehepatitis, familial adenomatous polyposis and ulcerative colitis.

The FcγRs can be divided into two general classes according to theirfunction which may be an activating or inhibitory one. The activatingreceptors are associated with a cytoplasmic 16 amino acid immunoreceptortyrosine-based activation motif (ITAM) having the consensus sequenceY-X₂-L/I-X₆₋₁₂-Y-X₂-I/L (Barrow and Trowsdale, EuJI, 2006, 36:1646-1653). This motif can be found, for example, in FcγRIIA. The otherclass of FcRs are inhibitory receptors which contain a 6-amino acidinhibitory motif (ITIM) in the cytoplasmic part of the receptor havingthe consensus sequence S/IN/L-X-Y-X₂-IN/L (Barrow and Trowsdale, EuJI,2006, 36: 1646-1653). An example of such an inhibitory FcR is FcγRIIB.

FcγRIIB (FcγRIIB) has two inhibitory activities. One of them isdependent on the ITIM-motif and occurs when FcγRIIB is ligated to anITAM-carrying receptor (e.g. FcγRIIA) resulting in the inhibition ofITAM-triggered calcium mobilization and cellular proliferation. Thesecond inhibitory action of FcγRIIB involves homo-aggregation of thereceptor (FcγRIIB clustering) which delivers a pro-apoptotic signal intothe cytoplasm. The pro-apoptotic signal has only been reported inB-cells and can be blocked by ligation of FcγRIIB to the B-cell receptor(BCR) (J V Ravetch, S. Bolland, Annu Rev. Immunol. 2001; 19:275-90.

As mentioned above, in WO 03/043648, SFcγRIIB has already been describedfor use in pharmaceutical preparations where relatively high amounts ofFcγ receptors can be included in a reasonable volume of a treatmentsolution for e.g. injection into a patient. Soluble Fcγ receptors andespecially SFcγRIIB has been suggested for the treatment of autoimmunediseases since they can bind to antibodies but do not affect othercomponents of the immune system. The soluble Fc receptors therefore areable to neutralize antibodies in the blood stream which has anattenuating effect especially on autoimmune processes. Possibleindications that are already mentioned in WO 03/043648 include solubleFcγ receptors for treatment of multiple sclerosis (MS), systemic lupuserythematosus (SLE) and rheumatoid arthritis (RA) and also for diseaseswith an elevated level of NK cells to avoid the disadvantages of thepreviously used treatment methods for such diseases.

The teaching of WO 03/043648 focuses on the therein discovered fact thatsoluble Fc receptors can be used to form aqueous solutions with aconcentration of up to 50 mg/ml of soluble receptor. For certainapplications such concentrations of active agent do suffice. In recentstudies, however, a therapeutic dose of sFcRs like SFcγRIIB ofsignificantly more than 1 mg/kg body weight of the patient has beenestablished to be beneficial or even necessary for a successfultreatment of autoimmune diseases.

Subcutaneous administration of pharmaceuticals is considered aneffective and relatively uncomplicated and non-onerous method ofdelivering an active agent to a patient. Compared to intravenousinfusion, which requires a more extensive medical equipment and in mostcases administration at a doctor's office or a clinic, subcutaneousadministration can easily be applied even by the patient himself.Subcutaneous administration will also result in a retarded onset ofaction, i.e. an increased half-life and time to maximal concentration.Also, it was found that the maximum plasma concentration is reduced incase of subcutaneous delivery. These effects are based on theadministration beneath the skin of a patient from where the active agentis transported to the bloodstream. Proteins larger than about 16-20 kDaare generally regarded to be taken up primarily by the lymphatic system,which might be an advantage for FcγRs as the target B cell populationmatures and resides in the lymphatics, too (Porter, C. J. H. andCharman, S. A. (2000), J. Pharm Sci. 89, 297-310).

The subcutaneous administration route, however, is preferably limited byan injection volume of 1.0 ml up to perhaps 1.5 ml per application(Gatlin, L. A. and Gatlin, C. A. B, (1999), Gapta, P. K. & Brazeau, G.A., eds., Interpharm Press, Denver, pp. 401-425). Thus, aqueous sFcγRsolutions as known from WO03/043648 could not have been considered forsubcutaneous administration. Rather, for sufficiently highconcentrations of receptor, precipitation and formation of undesirablylarge crystals of the receptors in the aqueous solution and accordinglyclogging of the needles and/or pain at the injection locus had to beexpected from the teaching of this document. Therefore, in spite of allthe anticipated advantages, subcutaneous administration didn't seem apromising route of treatment and rather intravenous injection orinfusion seemed to be the only viable delivery method.

Accordingly, it was one object of the present invention to provide meansand conditions for less complicated and burdensome administration of Fcreceptors to a patient and for aqueous formulations of soluble Fcreceptors, especially of soluble FcγRIIB, which contain the receptor ina sufficiently high concentration to enable a subcutaneous treatmentregimen for autoimmune diseases.

It was a further object of the present invention to provide such aqueousformulations of highly concentrated soluble Fc receptor in aready-to-use form which is stable under usual storage conditions forpharmaceuticals for more than 24 months or, alternatively, in a formwhich allows for long-term storage and easy and straightforwardadjustment and reconstitution prior to use in subcutaneous applications.

These objects were solved according to the present invention byformulations containing a soluble Fc receptor (sFcR) in an aqueousbuffered solution, wherein the concentration of the Fc receptor isgreater than 50 mg/ml and wherein it contains a physiologicallyacceptable buffer substance.

During the research which led to the present invention, it wassurprisingly found that, contrary to the previous expectations, it ispossible to provide soluble Fc receptors dissolved in suitable buffersolutions with a concentration of even much higher than 70 mg/ml andpreferably more than 150 mg/ml, by means of which it is for the firsttime possible to provide sFcRs also in the form of a pharmaceuticalcomposition for the subcutaneous application. The subcutaneousapplication as parenteral mode of application is simpler and fasterapplicable than an intravenous mode of application. As mentioned above,even the patient himself can carry out a subcutaneous application.

A short and usually thin cannula is required for subcutaneousadministration. The highly concentrated formulation of the presentinvention provides the sFcRs in a completely dissolved and homogenousform and at acceptable viscosity that permits the use of these thincannulas or as crystal suspension containing crystals that are smallenough to pass these thin cannulas. Thus, the patients' convenience isconsiderably increased and an administration of the receptors can beeffected by the subcutaneous route.

The inventive formulations as described in the following enable theprovision of soluble FcRs and especially of soluble FcγRIIB inconcentrations of up to a maximum value which is limited mainly by theincrease in viscosity of the solution due to the high concentration ofthe Fc receptor. Upon selection of appropriate buffer substances andadjustment of suitable osmolalities, a stabilization of 220 mg/mlSFcγRIIB and more at a physiological pH is enabled within the frameworkof the present invention.

The physiologically acceptable buffer substances, which are usedaccording to the present invention are commonly used buffers for thepharmaceutical application of suitable solutions. Within the frameworkof the present invention, however, it was found that depending on thebuffer substance used, the pH value of the solution has a considerableinfluence on the solubility of the Fc receptors. Depending on the bufferchosen, the pH value has to be adjusted within a determined range andespecially to a particular optimum for achieving the desired solubilityof the Fc receptors, especially if the purpose is to provide aformulation which does not contain any crystalline sFcR.

It was further found that the protein itself is able to sufficiently actas a buffer due to the presence of positively and negatively chargedamino acids. Thus, e.g. by selecting an appropriate pH value based onthe pK-value of the proteins amino acid side chains, it is possible toforgo the addition of a separate buffer substance as long as a suitableamount of protein is present in the solution to act as thephysiologically acceptable buffer substance.

In preferred embodiments of the present invention, as a buffersubstance, the formulation contains one of a histidine buffer, citratebuffer or phosphate buffer. However, it is particularly preferred toprepare the formulation with either a histidine or a citrate buffer. Inboth cases, it was found that the adjustment of the pH value in thesebuffered solutions permits an adaption of the solubility of the Fcreceptors by means of which high concentrations of Fc receptor can bedissolved, however, can also be caused to crystalllize by means ofincreasing or decreasing the pH value depending on the buffer substanceused. This possibility of changing between soluble and crystalline formsof sFcR by the mere adaption of the pH value implies considerableadvantages in view of adaptations with regard to the applicability,preservation and storage stability of pharmaceutical compositions.

For example, during marketing authorization procedures, sufficientstorage stabilities of pharmaceuticals have to be proven. In thiscontext, it is essential that stability data of a storage of at least 12months at a temperature of 5° C. be included by the party requesting themarketing authorization. However, it is desirable and advantageous thata storage stability for more than 24 months under correspondingconditions is achieved, whereby in view of the mentioned storageconditions, a substantial amount, preferably 90% of the pharmaceuticalagent still needs to be present in an active form after the expirationof the time.

In this context, the inventive formulations show particular advantages.For instance, they allow to offer highly concentrated sFcR formulationsin liquid form which exclusively contain dissolved Fc receptor and whichshow a high storage stability.

Moreover, the inventive formulations can be subjected to lyophilizationto provide a solid storage form. Such solid forms might even showimproved storage stability as compared to the liquid formulations. Suchformulations are therefore a further subject matter of the presentinvention.

Lyophilisation can be performed in any suitable manner known to theskilled person for lyophilising proteins. Preferably, conditions as mildas possible are used to avoid protein degradation.

From the solid form, ready-to-use liquid formulations can easily berestored by the addition of water for injection, saline or bufferedaequous solutions to provide the Fc receptor again in completelydissolved form or intermediate steps can be chosen in which thesolubility is adapted as desired.

Also concentrated forms of inventive formulations are a further subjectmatter of the present invention. Such concentrated forms can be obtainedby e.g. removing part of the liquid to below the solubility limit whichresults in formulations containing at least some crystalline receptors.Also from such formulations, the sFcR can be reconstituted to aready-to-use liquid formulation by adding water for injection, saline orbuffered aequous solutions to the desired concentration of active sFcRand especially to concentrations in which the sFcR is present entirelyin dissolved form.

Due to the pH dependency of the solubility of Fc receptor in theformulations according to the present invention, the formulationsprovide the additional advantage that the Fc receptor can practically becaused to crystallize entirely by means of adapting the pH and can beobtained (e.g. after sedimentation or centrifugation) as concentratedcrystal suspension. The crystals obtainable thereby can be very small(microcrystals), especially depending on the crystallisation conditions.The faster the crystallisation, the smaller the crystals are. Contraryto prior art, the present invention permits the fast and nearlyquantitative transformation of solubilised sFcR into proteinmicrocrystals and vice versa and thus allows to tailor the solubilityproperties of respective solutions and formulations. This is a furtherpossibility to ensure excellent storage stability and in particular theneed for only little storage space for an active agent, which can thenbe converted into an entirely or mainly soluble form by means ofdissolution in a suitable buffered aqueous solution having a suitable pHvalue. This can, for instance, be done immediately prior to itsapplication as a pharmaceutical by means of admixing concentratedcrystal suspension with a suitable buffer. Alternatively amicrocrystalline suspension or formulation might be directlyadministered by subcutaneous application as the present inventionprovides means to transform the receptor into microcrystals, i.e.crystals that are small enough to pass a cannula or thin needle.Compared to highly concentrated liquid formulations, the viscosity ofsuch microcrystal suspensions is much lower and does not riseexponentially with increasing protein concentration.

For the purposes of the present invention and as used above in thecontext of describing the present invention, Fc receptors are consideredas “crystalline” when crystals have an average size of more than 500 μmin diameter, whereas microcrystalline forms contain microcrystals with asize of equal to or less than 500 μm in diameter.

The present invention enables in an unprecedented way to provide solubleFc receptors in high concentrations in different forms suitable forimmediate or future pharmaceutical use. As illustrated above, this canbe effected in a ready-to-use dissolved form or in a solid, e.g.lyophilized form obtained from such solution or in microcrystalline formprecipitated by pH value adaption, which can then be reconstituted byresolubilization so that a formulation results in which the Fc receptoris contained in high concentration in a suitable buffer solution at thedesired pH value.

In preferred embodiments of the present invention, the inventiveformulation contains as an Fc receptor a sFcγ receptor. Regarding thepossibility of treating autoimmune diseases, the sFcγRII receptors andespecially SFcγRIIB have to be considered. A particularly preferredinventive formulation thus contains the soluble FcγRIIB receptor in apharmaceutically applicable solution with a suitable buffer substance.

For the purposes of the present invention, the FcγRIIB receptor has asequence as described in the prior art, especially WO 00/32767 and WO03/043648 or other documents referring especially to FcγRIIB andespecially SFcγRIIB. Further, the term is meant to encompass forms ofthe receptor which can differ especially in their N-terminal parts. Anespecially preferred SFcγRIIB protein is shown in SEQ ID NO:1. Thissequence contains a methionine residue at the N-terminus, which is e.g.required for prokaryotic expression, however is cleaved off in a majorpart of the produced proteins by bacterial mechanisms lateron. Thereforeproteins according to SEQ ID NO:1 lacking the N-terminal methionine arealso encompassed within the present invention as well as mixtures ofproteins with and without the N-terminal Met. Further, depending on theproduction process and the condition of the bacterial production strain,additional changes in the N-terminal five amino acids can occur. E.g. inaddition to methionine also the following residues can be cleaved off ormethionine could be exchanged for another amino acid like norleucine.Therefore also mixtures of all these proteins differing at theN-terminus and originating from production processes using a DNAsequence which encodes for the amino acid sequence of SEQ ID NO:1 areencompassed by the current definition of SFcγRIIB and especially by theterm huSFcγRIIB.

In further preferred embodiments, FcγRIIB proteins are considered asencompassed by the present invention as long as they have an at least90% identity to the protein of SEQ ID NO:1. For the determination ofsequence identity a comparison is made by aligning the sequences in amanner to provide the maximum correspondence of amino acids. It isespecially preferred that differences in the claimed proteins occur onlywithin the first ten and most preferably within the first five aminoacids. It is especially preferred that the proteins have an amino acididentity of at least 95% with differences occurring within the firstfive amino acids of SEQ ID NO:1, wherein the differences in the aminoacids are based on at least one of deletions, substitutions andadditions.

In a particularly preferred embodiment, this formulation according tothe present invention contains the sFc receptor and especially theSFcγRIIB receptor in concentrations of greater than 60 mg/ml, morepreferably greater than 60-80 mg/ml, still more preferably greater than80 mg/ml, even more preferably greater than 100 mg/ml and particularlypreferred greater than 150 mg/ml and most preferably even greater than200 mg/ml.

The inventive formulations optionally contain further pharmaceuticallyacceptable substances, which are for example used for the adjustment ofthe ionic strength of the solution and/or promote the solubility andstability of the receptor protein contained therein. Such substances areknown to the skilled person. For the adjustment of the ionic strengththe inventive formulation optionally contains a salt and preferablyNaCl. For the stabilization of the protein, polyols and especiallysugars and sugar alcohols like sucrose or mannitol can be used. Further,the inventive formulation preferably contains detergents which aresuitable for pharmaceutical applications, as for example polysorbates.

Buffer substances are preferably contained in the formulation of thepresent invention in an amount of 0.1 μM to 300 mM. In more preferredembodiments, the physiologically acceptable buffer is present in anamount of 0.1 to 150 mM and especially 1 to 50 mM.

For the adjustment of the osmolality (isotonicity), salts like sodiumchloride are suitable in such amounts which adjust a preferablyphysiological osmolality, at least as far as the formulation is intendedfor the direct administration to a patient. The osmolality of thesolution can be adjusted over a wide range and can be set to between 10mOsm/kg and greater than 600 mOsm/kg without having a considerableeffect on the solubility of the Fc receptor.

Salt, preferably NaCl, is present in the formulation in a concentrationof about 0 to 250 mM, preferably 5 to 200 mM and most preferably 10 to50 mM.

Polyols like sucrose are not necessarily contained in the inventiveformulations, however in preferred embodiments they are present in anamount of at least 1.0% and more preferred at least 2.0%. The preferredupper limit of the amount of polyols is approximately 25%, morepreferably 15% and most preferably 8%. Sugars are known to stabilizeproteins in solution.

Salts and sugars need to be balanced to adapt the osmolality of theformulation, preferably to be isotonic. The more sugar is contained inthe formulation, the less salt can be added and vice versa.

Suitable amounts of detergents, which are preferably used within thecontext of the invention, are 0.001-0.1%, particularly 0.005-0.05%.

As already mentioned above, the findings, which have been obtainedwithin the framework of the present invention, allow the adaption of thesolubility conditions for soluble Fc receptors and especially SFcγRIIBin such a way that for a content of Fc receptor of greater than 50mg/ml, a predetermined provision of the receptors is made possible ineither completely dissolved form or in a microcrystalline form foradministration to a patient. As also mentioned above, it is oftenadvantageous for an administration to a patient to provide as high aconcentration as possible in a completely dissolved form or at leasttransformable into a dissolved form.

For formulations containing microcrystalline sFcRs, an administration tothe patient may also be possible, whereby the microcrystals completelydissolve after administration and the active substance is available withits physiologic effect. For other administrations and also for storage,it can be of advantage to rather keep the receptor in crystalline form,wherein it is especially stable against degradation and thus loss ofactivity.

Accordingly, preferred embodiments of the present invention areformulations, which are completely liquid and wherein the receptor ispresent in solubilized form or in suitable microcrystalline form

An especially preferred formulation of the present invention containsthe soluble FcγRIIB receptor in a citrate buffered solution andpossesses a pH value of equal to or greater than 6. The pH value ispreferably adjusted within a range of 6.0-7.5. In such citrate bufferedsolutions with a physiological pH value, the FcγRIIB receptor is solublein concentrations of greater than 140 mg/ml. Due to the physiological pHvalue, such formulation also has the advantage that it can directly beadministered to a patient without causing side effects like pain at thesite of administration.

In another preferred embodiment, the soluble FcγRIIB receptor iscontained in a histidine buffered solution with a pH value of 5.2-5.9.When using a histidine buffer, SFcγRIIB is soluble in concentrations ofmore than 100 mg/ml.

At a pH of approximately 6.0, the solubility of the receptor is stillrelatively high, however, crystalline precipitates are beginning to formwhereas at a higher pH, only a substantially lower solubility of thereceptor is observed.

Both formulations described above enable a high concentration ofsolubilized SFcγRIIB receptor, whereby this could be shown for both thementioned citrate buffered formulation and for the histidine bufferedformulation up to the viscosity limited regimen of approximately 220mg/ml (see enclosed examples).

The inventive formulations further have excellent freeze/thaw stabilityproperties and also excellent stabilities at reduced temperatures of 2°C.-8° C. Even the stability at room temperature is excellent for thesesolutions.

The usability of both solutions is both given for a directadministration to the patient and in the production of a lyophilized orhighly concentrated formulation that can contain crystals and isconvenient for storing or generation of injection solutions which aredirectly reconstitutable by the patient.

For the direct administration, as already mentioned above, the citratebuffered solution with a physiological pH value is particularlypreferred.

A particularly preferred further subject of the invention is aformulation which contains the receptor in crystalline form. Suchformulations are preferably embodied as a citrate buffered suspensionwith a pH value of 5.2-5.9 or alternatively as a histidine bufferedsuspension at a pH value of 6.0-7.5. Such suspensions can for examplepreferably be used as storage-stable forms which, for the administrationto the patient, can be transformed to a formulation containing highconcentrations of the solubilized receptor by means of pH adjustment.Additionally, the same can also be concentrated or the receptor beobtained from them by separation of the solution in order to obtain ahighly concentrated crystal suspension. The receptor can be recovered incompletely dissolved form by reconstitution in a suitable buffer at asuitable pH value.

The described inventive formulations and the finding that, using certainbuffer substances, depending on the pH value different solubility levelsof Fc receptors can be realized enable on the one hand the provision ofready-to-use injections for the subcutaneous administration to apatient, or on the other hand the provision of particularlystorage-stable variants containing crystalline Fc receptor. Evenlyophilized or otherwise solid forms of the receptor are provided whichcan be transformed by a simple addition of the suitable solutions intoready-to-use forms containing high concentrations of soluble receptor.

A further subject of the present invention is thus a pharmaceuticalcomposition, which comprises a formulation according to the presentinvention as described above and in which further pharmaceuticallyacceptable excipients and/or adjuvants and/or carriers can be present.In particularly preferred embodiments these pharmaceutical compositionsare directly applicable for the subcutaneous injection of an effectiveamount of a soluble Fc receptor and especially for the treatment ofautoimmune diseases.

In one preferred embodiment, the pharmaceutical composition preferablycontains a sufficient amount of completely dissolved sFc receptor in asuitable buffer substance and at a physiological pH value. Suchpharmaceutical composition is a ready-to-use medicament and can bedirectly applied to the patient. The dissolved soluble receptor can e.g.easily be absorbed into the patient's lymphatic circulation and bedirectly effective there or after transport within the patient's body byblood or body fluid circulation.

Alternatively, the pharmaceutical composition can contain the receptorin a highly concentrated and at least partially microcrystalline orcrystalline form. Diluted as necessary with suitable buffer solutions,the pharmaceutical composition is again particularly suitable for thesubcutaneous injection of an effective amount of Fc receptors.

As already explained above in the context of describing the presentinvention, Fc receptors are considered as “crystalline” when crystalshave an average size of more than 500 μm in diameter, whereasmicrocrystalline forms contain crystals with a size of equal to or lessthan 500 μm in diameter. As far as a direct application to the patientis considered, pharmaceutical compositions containing completelydissolved Fc receptor can of course be used but also pharmaceuticalcompositions containing formulations with the Fc receptor being solelyor partially in microcrystalline form have merit in pharmaceuticalapplications. These microcrystals are small enough to not clog theneedles for subcutaneous application. The use ofmicrocrystals-containing solutions can be beneficial for e.g. delayed orsustained release of the active sFc receptor to the patient's systemand, under certain circumstances, such microcrystalline forms can evenbe considered as preferred pharmaceutical compositions.

Pharmaceutical compositions of the present invention containingmicrocrystalline or crystalline forms of Fc receptors can e.g. beobtained by concentration of the sFc beyond its solubility byconventional concentration techniques like ultrafiltration. It ispossible to maintain the pharmaceutical in liquid form containing acertain amount of crystals or microcrystals. Instead of using amechanical concentration method for obtaining crystals or microcrystals,it is also possible and a preferred embodiment of the present inventionto crystallize the receptor by adjusting the pH to a value wherein thereceptor has a considerably lower solubility. The precipitated crystalsor microcrystals can be separated from the solution and used for storageand subsequent reconstitution or direct administration. The solid formsof receptor formulations are especially storage stable and maintaintheir effectiveness for at least more than 24 months.

In such cases, the pharmaceutical composition is conveniently providedin a pharmaceutical kit format, which in addition to the solid or highlyconcentrated sFcR also comprises the suitable liquid for thereconstitution of an injectable solution. A further subject matter ofthe present invention is therefore a pharmaceutical kit containingcrystalline or lyophilized soluble Fc receptor and a suitablepharmaceutically acceptable liquid like buffer solution or simply waterfor the reconstitution of the injection solution in suitable separatestorage units.

It is especially preferred for an inventive pharmaceutical kit, if sFcRreceptor and buffer solution for the reconstitution of the injectionsolution are provided in suitable devices for simple mixing and wellprotected against contamination. Preferably, the kit contains a buffersolution based on a phosphate buffer, a histidine buffer or a citratebuffer. It is further preferred for the buffer solutions that the pH isadapted to provide optimum solubility for the Fc receptor. In especiallypreferred embodiments of the present invention, the buffer solution is acitrate buffered solution with a pH of above 6, especially 6.1 to 7.5,or a histidine buffered solution with a pH of below 6.0, especially 5.2to 5.9. The citrate buffered solution is the most preferred buffercontained in a pharmaceutical kit according to the present invention.

The amount of buffered solution contained within the pharmaceutical kitis adapted to the amount of solid or concentrated sFcR in the kit.Depending on whether a complete dissolution of the sFc receptor ormaintaining some amount of microcrystals is desired, a correspondingbuffer is selected in a suitable amount of liquid and also the pH of thesolution is adapted according to the teaching concerning sFcR solubilityas provided herein.

A further subject matter of the present invention is the use of theformulations, the pharmaceutical compositions and pharmaceutical kits ofthe present invention for the prevention or treatment of autoimmunediseases. More particularly, the present invention is intended for theuse within the framework of the prevention or treatment of multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis, primaryimmune thrombocytopenia and autoimmune haemolytic anemia (AIHA).Further, the formulations, pharmaceutical agents and pharmaceutical kitscan be used for the treatment of inflammatory disorders. The inventivesubject matter enable the application of Fc receptors for allindications for which these have already been described or for whichthey are considered to be suitable in the future. The inventiveformulations and pharmaceutical compositions and kits allow especiallythe subcutaneous administration which is very efficient and easilyapplicable to (or by) a patient. The possibility of administeringespecially high amounts and concentrations of Fc receptors is aparticular advantage of the present invention.

The following examples shall further explain the invention and itsadvantageous effects and embodiments.

Example: Development of High Concentrated Liquid husFcγRIIb FormulationsSuitable for Subcutaneous Delivery 1. Materials

The following solutions of huSFcγRIIB (soluble human FcγRIIB receptorhaving the amino acid sequence as shown in SEQ ID NO. 1) were used asparent material for all experiments:

a) husFcγRIIB 5 mg/ml concentrate for solution for infusion

-   -   5 mg/mL husFcγRIIB in 5.3 mM NaH₂PO₄, 1.94 mM KH₂PO₄, 150 mM        NaCl, 2% (w/v) mannitol, 0.005% polysorbate 20 pH 6.5

b) husFcγRIIB 20 mg/mL concentrate for solution for infusion

-   -   20 mg/mL husFcγRIIB in 20 mM histidine, 150 mM NaCl, 2% (w/v)        sucrose, 1% (w/v) mannitol, 0.005% polysorbate 20 pH 6.5.

The following chemicals of at least the indicated grades were used:

Name Purity Supplier Citric acid monohydrate p.a. Merck Sodium hydroxidePh. Eur. (≥98%) Carl Roth Sodium chloride Ph. Eur. (≥99%) Carl RothEthanol, abs. Ph. Eur. (≥99.8%) Carl Roth Histidine Ph. Eur. (≥98.5%)Carl Roth Hydrochloric acid, 37% p.a. Carl Roth Sucrose Ph. Eur. (≥99%)Carl Roth Mannitol ACS reagent (≥99%) Fluka Polysorbate 20 (Tween 20)cell culture tested Sigma Aldrich Trehalose for biochemistry Merck

2. Methods

a) husFcγRIIB Content by UV/Vis Spectroscopy

The sample was transferred to a UV micro-cuvette (UV cuvette micro,Plastibrand, Brand) and the absorbance was measured with aSpectrophotometer (Cary 100, Varian) using the respective buffer asblank. The huSFcγRIIB concentration was calculated by the followingequation:

huSFcγRIIBconc. [mg/mL]=(A₂₈₀−A₃₂₀)×DF×0.64 DF≡dilution factor

b) husFcγRIIB Buffer Exchange by Cation Exchange Chromatography

1000-1800 mg huSFcγRIIB (huSFcγRIIB 5 mg/mL concentrate for solution forInfusion) was carefully diluted with 10 mM citrate/NaOH pH 6.5 until theconductivity reached 5.0±0.1 mS/cm. The diluted protein was filtered(0.2 μm Durapore membrane PVDF hydrophil, 47 mm, Millipore) and loadedat 5.5 mL/min onto a 57 mL SP Sepharose HP cation exchange (CEX) resin(26×107 mm, GE Healthcare; equals 17.5-31.6 mg huSFcγRIIB/mL resin)equilibrated in 10 mM citrate/NaOH, 20 mM NaCl pH 6.5. Bound protein waswashed at 5.5 mL/min with 200 mL 10 mM citrate/NaOH, 20 mM NaCl pH 6.5and eluted with a 300 mL linear gradient ranging from 20 mM to 600 mMNaCl in 10 mM citrate/NaOH pH 6.5. Eluate collection was started afterOD₂₈₀ exceeded 250 mAU (AU: adsorbance units) and was stopped after itdropped below 200 mAU (1 cm path length, Äkta Explorer 100, GEHealthcare). The column was regenerated with 100 mL 1 M NaCl, washedwith 150 mL MilliQ H₂O (ultrapure water, Millipore Corp.) and storeduntil further use in 20% ethanol. After huSFcγRIIB content measurementby UV/Vis spectroscopy the eluate was filtered (Millex 33 mm, 0.2 μmDurapore PVDF (polyvinyliden fluoride) hydrophil, both from MilliporeCorp.), aliquoted, snap-frozen in liquid nitrogen and stored at ≤−70° C.until use.

The NaCl content was calculated by correlation of the mean conductivityof the eluate to the measured conductivity at 10 mM citrate/NaOH, 20 mMNaCl and 10 mM citrate/NaOH, 600 mM NaCl.

Histidine buffered huSFcγRIIB was prepared similarly using 10 mMhistidine/HCl as buffering species.

c) pH Stability Screen

A concentrated huSFcγRIIB solution in 20 mM histidine, 324 mM NaCl pH6.5 was adjusted to 0.5 mg/mL huSFcγRIIB, 20 mM histidine, 150 mM NaCl,2.5× Sypro Orange (5000× in DMSO, Molecular Probes™, Invitrogen) usingsuitable stock solutions. The pH of the solution was adjusted between pH4 and pH 12 based on an experimental titration curve with 0.2 M HCl or0.2 M NaOH. 40 μL of the solution were incubated in a sealed 96 wellhalf area well plate (pclear , black, medium binding; Greiner BioOne)for 3 h at 25° C. and assayed for Sypro Orange fluorescence (Excitation485 nm, Emission 590 nm, gain 60, lag time 0 μs, integration time 40 μs;TECAN Spectrofluor plus).

d) Preparation of High Concentrated Formulations

The required NaCl content of histidine or citrate buffered huSFcγRIIB(initial concentration approx. 200-300 mM NaCl) was adjusted by dilutionwith the appropriate buffer (10 mM citrate or 10 mM histidine pH 6.5)and subsequent ultrafiltration (Vivaspin 20, 5 kDa MWCO, Sartorius). Inorder to keep the processed volumes small, the procedure was repeatedfor up to 3 cycles in total. After final dilution, the pH was measured(pH-meter HI8314, pH-electrode HI1217, Hanna instruments), adjusted with0.2 M NaOH or 0.2 M HCl in the appropriate buffer and the proteinsolution was concentrated. The huSFcγRIIB content was measured by meansof UV/Vis spectroscopy in triplicate, the huSFcγRIIB concentration wasadjusted with the appropriate buffer and the solution was filtered(Ultrafree MC, 0.2 μm Durapore PVDF hydrophil, Millipore).

e) husFcγRIIB Solubility Screens in 384 Well Format

The solubility of high concentrated huSFcγRIIB solutions in relation tobuffering species, pH, salt concentration and sugar/polyol concentrationwas assessed in 384 well format (pclear, white, non-binding, GreinerBioOne) using 30 μL per well. The final formulations were prepared bydirect addition of filtered stock solutions to each well. The followingstock solutions were used: 100-195 mg/mL huSFcγRIIB, 1.5 M NaCl and 30%(w/v) sucrose+15% (w/v) mannitol in either 10 mM citrate pH 7.0 or 10 mMhistidine pH 5.5. Each solution was supplied with 0.01% (w/w) polysorbat20 and depending on the screen with 10-50 mM NaCl.

The pH of each well was adjusted with 0.5 M-0.75 M HCl or NaOH. Therequired amount of acid or base was calculated based on theoreticaltitration curves assuming that all nine histidine residues of huSFcγRIIB(pK_(a)=6.00) provide additional buffering capacity. The plate wascentrifuged (500·g, 1 min), sealed with adhesive tape (microtest tape,permacel, neo-lab) and stored at 5±3° C. in the dark.

The visual appearance of each formulation was assessed by lightmicroscopy (Axiovert 25f, Carl Zeiss) and ranked according to anarbitrary scale (0=no crystals; 1=some crystals, hardly visible; 2=somecrystals clearly visible; 3=more than 30 crystals per well clearlyvisible; 4=incomplete layer of many crystals (well not fully covered);5=full layer of many crystals (well completely covered).

Taken into account dissolved salt, sugar and protein the osmolality ofeach formulation was calculated according to the following equation:

$\underset{i}{\xi_{m}} = {\Sigma v_{i}m_{i}F_{m,i}}$

wherein v_(i) is the number of particles formed by the dissociation ofone molecule of the i^(th) solute and m_(i) is the molality of the i″solute. For simplicity the molal osmotic coefficient F_(m,i) for eachsolute was assumed to be equal to 1.

f) Small Scale Crystallization of husFcγRIIB

10 μL-450 μL husFcγRIIB at 50-140 mg/mL in 10 mM histidine, 10 mM NaCl,0.01% polysorbate 20, pH 5.5 were diluted with appropriate diluents to40 mg/mL husFcγRIIB in 10 mM histidine, 10 mM NaCl, 0.01% polysorbate 20in a 1.5 mL polypropylene reaction tube. The pH was adjusted to 6.5-7.2by the addition of 4.38-6.23 Vol % (final volume after addition ofdiluents) 0.3 M NaOH. The required amount of acid or base was calculatedbased on theoretical titration curves assuming that all nine histidineresidues of husFcγRIIB (pK_(a)=6.00) provide additional bufferingcapacity.

g) Differential Scanning Fluorometry

120 μL of each formulation containing 0.5 mg/mL husFcγRIIB were preparedin a 1.5 mL test tube similarly to the procedure described above in2.e). Sypro Orange (5000× in DMSO, Molecular Probes™, Invitrogen) wasadded to a final concentration of 2.5× using a 200× stock in theappropriate buffer. 30 μL of each formulation were transferred intriplicate to a well plate (MicroAmp 96 well optical reaction plate,Applied Biosystems) and the plate was sealed with adhesive tape(MicroAmp optical adhesive film, Applied Biosystems). The plate wassubjected to a temperature ramp from 19° C. to 90° C. with a slope of 1°C./min and the fluorescence emission at 610 nm was recorded (7300 RealTime PCR system, Applied Biosystems). The fluorescence wasdifferentiated with respect to time, a spline was calculated and thefirst detected maximum was reported as the melting temperature ofhusFcγRIIB (Origin 8.0, OriginLab).

h) Turbidity Screen in 384 Well Format

husFcγRIIB formulations were prepared in a 1.5 mL test tube similarly tothe procedure described above in 2.3). 30 μL of each formulation weretransferred in duplicate to a 384 well plate (pclear, white,non-binding, Greiner), the plate was sealed with adhesive tape(microtest tape, permacel, neo-lab) and placed in an incubator. Ascontrol the respective placebo solutions were prepared. The turbiditywas measured at 360 nm (Spectrafluor, bandpass filter 360/35 nm, 3flashes, Tecan). To avoid corrupted measurements due to the condensationof water, the plate reader was pre-heated to the assay temperature.

i) Dynamic Viscosity by Pressure Drop Measurement

The dynamic viscosity of huSFcγRIIB containing formulations wasdetermined by measuring the pressure drop as liquid flows through a flowchannel (m-Vroc, Rheosense). For each measurement 100 μL containingformulation was filled with a 200 μL pipette into the cylinder of a 100μL gastight syringe (Hamilton). The syringe was installed into therheometer and 80 μL were injected at a flow rate of 50 μL/min and 20° C.

j) Quantitative Polysorbat 20 Assay

The polysorbate content was determined by a modified protocol which isbased on the colorimetric assay first described by Brown and Hayes,(1955) Analyst 80, 755-767. 500 μL of the solution to be analysed wereextracted three times with 500 μL ethylacetate in a 1.5 mL polypropylenetube (VWR). To accelerate the phase separation, the tube was centrifuged(20,000·g, 5 min, 25° C.). The organic supernatants were combined in aHPLC vial (ND9, screw threaded, with conical bottom and PTFE screw cap,VWR) and the solvent was evaporated (25° C., 10 mbar, 0.5 h-1 h). Theresidual solids were suspended in 800 μL reagent solution (100 mMCo(NO₃)₂, 2.63 M NH₄SCN in water) and were extracted with 150 μL CHCl₃.100 μL of the CHCl₃ extract were transferred to a quartz UV microcuvette (Helima), the spectrum was measured from 200-800 nm (8453 diodearray spectrophotometer, Agilent) and the absorbance at 620 nm correctedby the absorbance at 530 nm was recorded. As blank an extract of anequivalent solution containing no polysorbate 20 was used. Each samplewas prepared in duplicate. The polysorbate 20 content was determinedbased on a standard curve from 0 to 0.006% (w/w) polysorbate 20 in therespective buffer.

k) Lyophilisation

59 formulations containing 15 -120 mg/mL huSFcγRIIB in 5 mM citrate,10-25 mM NaCl, 2-8%(w/v) sucrose, trehalose or mannitol and 0.005-0.01%(w/v) polysorbate 20 were prepared and 400 μL were filled into 1.5 mLclear HPLC vials (32×11.6 mm, wide opening, VWR). The vials weresubjected to a conservative lyophilisation cycle using the freeze-drierEpsilon 2-12D FD02 (Martin Christ, Osterrode, Germany). The vacuumduring the freeze-drying process was controlled by a MKS CapacitanceManometer. The samples were frozen at −45° C., primary drying wasperformed for 15 h at 45° C. to 15° C., 0.12 mbar and secondary dryingwas performed for 10 h at 15° C. to 20° C., 0.12 mbar. The lyophilisatewas reconstituted in 100-400 μL water for injection. The solution wasanalyzed in respect to the formation of particulates by visualinspection, turbidity measurement and fluorescence microscopy. In brief,for fluorescence microscopic examination 50 μL of the huSFcγRIIBcontaining solution was placed into a 384 well plate (pclear, white,non-binding, Greiner) and mixed with 5 μL 25× Sypro Orange (5000× inDMSO, Molecular Probes™, Invitrogen) in 5 mM citrate, 10 mM NaCl pH 6.7.The plate was incubated for 10 min at 25° C., centrifuged (1,000·g, 3min) and the appearance of each formulation was assessed by fluorescencemicroscopy (Axiovert 25f, excitation filter 470/20 nm, dichroic 493 nm,emission filter 503-530 nm, Carl Zeiss).

3. Results

a) Definition of pH Range and Buffering Species for High ConcentratedhusFcγRIIB Solutions The huSFcγRIIB denaturing pH range was determinedusing Sypro Orange as an indicator for the presence of unfolded protein.Sypro Orange is an environment sensitive dye whose fluorescence emissionis strongly increased after its binding to hydrophobic structures(Layton & Hellinga, 2010, Biochemistry 49 (51), 10831-10841). FIG. 1shows the results of experiments to determine the denaturing pH range.Therefore, 0.5 mg/mL huSFcγRIIB in 20 mM histidine, 150 mM NaCl (o) andblank buffer (+) were incubated at the respective pH for 3 h at roomtemperature. An increase in Sypro Orange fluorescence indicated thepresence of denatured huSFcγRIIB. As shown in FIG. 1, huSFcγRIIB did notunfold from pH 5.2 to at least pH 11.

To prevent pain during subcutaneous administration the administeredsolution should have a pH in the physiologic range. Typical bufferingspecies that buffer in this range and are generally regarded as safeinclude histidine (pka ˜6.0), citrate (pKa₃ ˜6.4) and phosphate (pKa₂˜7.2). Due to its tendency to promote pH shifts during freeze/thaw(MacKenzie, 1977) phosphate was not included in subsequent solubilityscreens.

In an initial attempt to determine the limiting huSFcγRIIB concentrationin respect to protein precipitation huSFcγRIIB was concentrated byultrafiltration in the presence of 10 mM histidine or 10 mM citrate and10 mM NaCl until a visible precipitate was formed. As shown in Table 1histidine buffered huSFcγRIIB showed increased solubility in theslightly acidic range from pH 5.5 to 6.0 whereas citrate as bufferingspecies provided solubility in the near neutral pH range around pH 6.5.In summary the huSFcγRIIB solubility at various pH is largely dependenton the buffering species used. By microscopy it was shown that theprecipitate is composed of crystalline needles.

TABLE 1 husFcγRIIB solubility limit (in mg/mL) in 10 mM histidine or 10mM citrate at low ionic strength from pH 5.5 to 7.5. husFcγRIIB wasconcentrated by ultrafiltration until the solution became cloudy.Histidine buffered husFcγRIIB precipitated at pH 6.0, 6.5 and 7.0whereas citrate buffered husFcγRIIB precipitated at pH 5.5 and pH 6.0.The sediment was identified as husFcγRIIB protein crystals. pH 5.5 6.06.5 7.0 Histidine >162 ≤174 ≤40 ≤10 Citrate ≤40 ≤158 >154 >148

b) Crystallization of husFcγRIIB Histidine buffered husFcγRIIB remainssoluble above 100 mg/mL at pH 5.5 and can be crystallized byneutralization at low ionic strength, on the contrary citrate bufferedhuSFcγRIIB remains soluble at neutral pH above 100 mg/mL and can becrystallized by mild acidification at low ionic strength (Table 1). In afurther test, crystallisation of huSFcγRIIB was investigated independence of the presence and the amount of sugar and NaCl in thebuffer. huSFcγRIIB crystallisation was performed in 10 mM histidine pH6.7 (FIG. 2a ) or 10 mM citrate pH 5.5 (FIG. 2b ) as a function of NaCland sugar (2:1 sucrose:mannitol) concentration. huSFcγRIIB in 10 mMhistidine, 10 mM NaCl pH 5.5 or 10 mM citrate, 10 mM NaCl pH 7.0,respectively, was concentrated to 140 mg/mL by ultrafiltration anddiluted to 40 mg/mL with appropriate stock solutions. In case ofhistidine buffered huSFcγRIIB, the crystal yield was determined bymeasuring the huSFcγRIIB concentration in the supernatant after 3 d at2-8° C. In case of citrate buffered huSFcγRIIB, no crystal growth wasdetected until day 10. Therefore, the crystal yield was determined after14 d at 2.8° C. Each solution contained 0.01% polysorbate 20.

FIG. 2 shows that huSFcγRIIB crystallizes more readily and the crystalyield is much higher in the presence of 10 mM histidine, 10 mM NaCl pH6.7 compared to 10 mM citrate, 10 mM NaCl pH 5.5. Further reduction ofthe sodium chloride concentration from 10 mM to 5 mM resulted in amarginal increase in crystal yield when using histidine as bufferingspecies but showed a strong effect when using citrate buffer. Reductionof the salt concentration below 5 mM and/or reduction of pH mightfurther stimulate the crystallization process in the presence ofcitrate. Increased NaCl concentrations or the addition of polyols, e.g.sucrose or mannitol, above 5% inhibited the crystallization process.

FIG. 3 shows an experiment in which huSFcγRIIB crystallisation wasperformed in 10 mM histidine, 10 mM NaCl as a function of pH. huSFcγRIIBin 10 mM hisitidine, 10 mM NaCl pH 5.5 was concentrated to 140 mg/mL byultrafiltration and diluted to 40 mg/mL with appropriate stocksolutions. The crystal yield was determined by measuring the huSFcγRIIBconcentration in the supernatant after 3d at 2.8′° C. Each solutioncontained 0.01% polysorbate 20.

Within a pH range of at least 0.5 units, more than 93% of histidinebuffered huSFcγRIIB can be crystallized. With a total huSFcγRIIBconcentration of 40 mg/mL the solubility limit of huSFcγRIIB in 10 mMhistidine, 10 mM NaCl pH 6.7-7.2 is below 2.8 mg/mL. At pH 6.9 thecrystallization was complete in less than one hour at 25° C.

c) husFcγRIIB Solubility Screens

In order to define so called solubility sweet spots, i.e. conditionswhere huSFcγRIIB remains soluble above 100 mg/mL and does notcrystallize, various huSFcγRIIB formulations were prepared in a 384 wellmicrotiter plate and incubated for at least 4 weeks at 2-8° C.Parameters included into the screen were huSFcγRIIB concentration (70,100, 120, and 150 mg/mL), buffering species (histidine or citrate), pH(5.5, 6.0, 6.5, 7.0, 7.5), NaCl concentration (10-225 mM) and sugarcontent (0-7.5%). The initial screens at 70 mg/mL (Table 2 and Table 5)and 100 mg/mL huSFcγRIIB (Table 3 and Table 6) were conducted with twosugar levels (0% or 3%) and four different NaCl concentrations (10, 50,225 mM).

Basically these prescreens reproduced the results that were alreadyobtained during the above mentioned concentration screen (Example 2a)and crystallization screens (Example 2b). Histidine buffered husFcγRIIBcrystallizes above pH 5.5, whereas citrate buffered huSFcγRIIBcrystallizes at pH 5.5 to 6.0. Increasing NaCl or sugar concentrationsreduced the crystallization process in both cases.

In all subsequent screens at 120 mg/mL and 150 mg/mL huSFcγRIIB, onlyformulations that are isotonic with blood, i.e. with a calculatedosmolality around 308 mOsmol/kg were included. For that reason highsugar concentrations were matched with low concentrations of NaCl andvice versa. Histidine based formulations with 120 mg/mL huSFcγRIIB wereable to prevent crystallization of huSFcγRIIB under neutral pH and highionic strength but with one exception all histidine based formulationsat 150 mg/mL huSFcγRIIB showed strong crystal growth after 4 weeks at2-8° C. (Table 4). On the other hand, citrate based formulations werestable up to 150 mg/mL huSFcγRIIB from pH 6.5 to 7.5 and at allsugar/salt combinations tested (Table 7).

Therefore citrate buffered huSFcγRIIB represents the best basis for thedevelopment of a high concentrated liquid formulation suitable forsubcutaneous application, i.e. with physiologic pH and tonicity.

d) Thermal Stability of High Concentrated husFcγRIIB Formulations

The formation of non-native protein aggregates and particulates couldpose a major obstacle for the development of a high concentrated proteinformulation (Shire et al., 2010, Chapter 15. High-concentration antibodyformulations. In Formulation and Process Development Strategies forManufacturing Biopharmaceuticals. Jameel, F. & Hershenson, S., eds.,John Wiley, Hoboken, N.J.). For that reason formulation candidates (c.f.Example 2c) were ranked in respect to their ability to preservehuSFcγRIIB's native structure in the presence of thermal stress andhence prohibit non-native aggregation.

At first the melting temperature T_(m) of citrate buffered huSFcγRIIBformulations was measured by Differential Scanning Fluorimetry. FIG. 4shows the respective results that were obtained using 0.5 mg/mLhuSFcγRIIB in 10 mM citrate, 4.5% sugar (2:1 (w/w) sucrose:mannitol), 75mM NaCl at the indicated pH (FIGS. 4 (a) and (c)), or 0.5 mg/mLhusFcγRIIB in 10 mM citrate pH 7.0 supplemented with the indicatedamount of sugar (2:1 (w/w) sucrose:mannitol) and salt (FIGS. 4 (b) and(d)). huSFcγRIIB formulations were heated in a 96 well microtiter plateat 1° C./min in the presence of Sypro Orange and the fluorescenceemission at 610 nm was recorded. The fluoresence vs. temperature plots(a) and (b) and their first derivatives (c) and (d) are shown. The firstmaximum in the dF/dT plots was defined as huSFcγRIIB meltingtemperature.

Depending on the composition of the respective formulation candidateT_(m) values from 50.8° C. to 55.5° C. were measured. The largestinfluence on the melting temperature had the pH with a T_(m) increase byapprox. 3.5° C. when the pH was lowered from 7.5 to 6.5. Addition of7.5% sugar increased the T_(m) by approx. 1° C. (FIG. 5, showing thechange of the huSFcγRIIB melting temperature as a function of pH, sugarand salt concentration in 10 mM citrate. The average and standarddeviation from three independent wells are shown). Although the NaClconcentration was lowered in parallel the increased thermal stability isclearly a function of the increased sugar concentration and not thedecreased salt content, as shown in earlier experiments (data not shown)and according to the theory of preferential exclusion (Timasheff, 1992,Chapter 9. Stabilization of Protein Structure. In Stability of ProteinPharmaceuticals, Part B: In vivo pathways of degradation and strategiesfor protein stabilization. Ahern, T. J. & Manning, M. C., eds., PlenumPress, New York, pp. 265-285). This is also exemplified by raising thesugar concentration from 7.5% to 40% which resulted in a T_(m) increaseby 6.2° C.

Next it was determined to what extent the stabilization of huSFcγRIIB'ssecondary and tertiary structure, as indicated by a high meltingtemperature, would inhibit the formation of insoluble proteinaggregates. Therefore the turbidity of citrate buffered huSFcγRIIBformulations was measured after incubation at 37° C., a temperature wellbelow the measured T_(m). To this purpose, the accelerated stability ofcitrate buffered huSFcγRIIB formulations at 37° C. were determined. Theoptical density at 360 nm was measured as a function of huSFcγRIIBconcentration and sucrose concentration after 1 h (a), 12 h (b) and 7 d(c). All formulations contained 10 mM citrate pH 7.0, 25 mM NaCl. Theoptical density of a buffer control was subtracted. At 40% sucrose, thehighest concentrated formulation contained only 60 mg/mL husFcγRIIB andnot 80 mg/mL.

The results are shown in FIG. 6. With increasing sucrose concentration(increasing concentrations from back to front on z-axis in all diagramsof FIG. 6), i.e. increasing melting temperature of the protein, the risein turbidity is retarded. With no added sucrose all formulations with astrength 0 mg/mL huSFcγRIIB became already turbid after one hour at 37°C., whereas at 40% sucrose no significant increase in turbidity up to 60mg/mL huSFcγRIIB was observed even after seven days at 37° C. Above acertain protein concentration the absolute turbidity increases linearlywith increasing huSFcγRIIB concentration but below that threshold theformation of insoluble protein aggregates is extremely slow or eveninhibited. With increasing sucrose concentrations this threshold isshifted to higher huSFcγRIIB concentrations, but from the physiologicpoint of view unacceptable high sucrose concentrations will be needed tostabilize huSFcγRIIB at 37° C. and a concentration exceeding 60 mg/mL.

A further test was conducted regarding the accelerated stability ofcitrate buffered huSFcγRIIB formulations at 40° C. The results are shownin FIG. 7. The increase in optical density at 360 nm was measured at 10mg/mL huSFcγRIIB in 10 mM citrate, 150 mM NaCl pH 7.0 supplemented with10%/292 mM sucrose (a, Δ), 10%/292 mM trehalose (a, □), 5%/274 mMmannitol (a, ◯), 30%/876 mM sucrose (b, Δ), 30%/876 mM trehalose (b, □),15%/822 mM mannitol (b, ◯). The buffer control supplemented with 20%sucrose is also shown (⋅).

Based on these observations different sugars and sugar alcohols wereranked in respect to their ability to suppress the formation ofinsoluble protein aggregates. As shown in FIG. 7, the most efficientstabilizer is sucrose.

e) Definition of Required Detergent Concentration

Turbidity assays (data not shown) indicated in accordance with publisheddata (Timasheff, 1992, Chapter 9. Stabilization of Protein Structure. InStability of Protein Pharmaceuticals, Part B: In vivo pathways ofdegradation and strategies for protein stabilization. Ahern, T. J. &Manning, M. C., eds., Plenum Press, New York, pp. 265-285) thatincreasing detergent concentrations destabilize husFcγRIIB. Also it isspeculated that the use of high detergent concentrations may lead toincreased immunogenicity (Hermeling et al., 2003, Pharm. Res. 20,1903-1907). For that reason it would be mandatory to keep the detergentconcentration as low as possible without compromising its stabilizingeffect towards surface stress.

Ideally, the polysorbate 20 concentration would be fixed at 0.005%, theconcentration which is used for already established liquid husFcγRIIBformulations containing 5-20 mg/mL husFcγRIIB. But since the strength ofthe newly developed formulation will be above 50 mg/mL and polysorbate20 may bind to the protein, it was questioned whether the detergentconcentration must be raised above 0.005%.

In order to test the hypothesis of unspecific detergent binding by theprotein a citrate buffered formulation candidate containing 0.005%polysorbate 20 was supplied with increasing concentrations of husFcγRIIBand the solutions were incubated for one hour at room temperature and60° C., a temperature well above the determined T_(m) of husFcγRIIB.After the protein and hypothetically bound polysorbate 20 were removedby cation exchange chromatography the amount of free polysorbate wasmeasured. In FIG. 8 an experiment is shown that determines freepolysorbate 20 in the presence of husFcγRIIB. husFcγRIIB in 10 mMcitrate, 25 mM NaCl, 3% sucrose, 1.5% mannitol, 0.005% polysorbate 20,pH 6.7 was incubated for 1 h at 25° C. (native huSFcγRIIB) and 60° C.(denatured husFcγRIIB). After husFcγRIIB was removed by cation exchangechromatography (CEX), the amount of polysorbate 20 was measured (a). Theblank represents buffer without added husFcγRIIB or detergent. Thesystem suitability tests (SST) represent buffer with 0.005% polysorbate20, SST2 was CEX treated and SST1 not. The measured polysorbate contentat 0.08-0.72 mg/mL husFcγRIIB was extrapolated to husFc∛RIIBconcentrations above 10 mg/mL by linear regression (R²>0.998) of thepolysorbate concentration versus the logarithmic husFcγRIIBconcentration (b).

As shown in FIG. 8, a linear relationship between the free polysorbatecontent and the logarithmic husFcγRIIB concentration was established.Therefore it is expected that the concentration of free polysorbate willonly change marginally, by approx. 0.0001-0.0002%, in case that thestrength of the formulation is increased from 20 to 100 mg/mL. Thesignificantly lower polysorbate concentration in the samples compared tothe control at 0.005% is largely attributed to the fact the samples werediluted with CEX equilibration buffer, which was prepared withoutpolysorbate 20, during column loading (c.f. FIG. 8 SST 1 vs. SST2).

f) Viscosity of High Concentrated husFcγRIIB Formulations

Highly concentrated protein formulations are characterized by a highviscosity (Shire et al., 2010, supra). Therefore the manufacturabilityof a highly concentrated protein formulation might be hampered by itsviscosity as the concentration process by tangential flow filtration maybecome unacceptable slow. In a further experiment, the solutionviscosity of husFcγRIIB in 10 mM citrate, 25 mM NaCl, pH 7.0 wasmeasured at 20° C. The experimental data as shown in FIG. 9 (◯) wasfitted to an exponential growth function (−, R²>0.992) and found thatthe solution viscosity of the formulation is low enough to run economicTFF processes up to at least 210 mg/mL.

g) Lyophilisation of husFcγRIIB Containing Formulations

59 formulations with varying husFcγRIIB, sugar, salt and detergentcontent were subjected to a conservative lyophilisation cycle. Thesolids were reconstituted with a volume of water for injection that wasequal to or less than the original volume prior to the lyophilisationprocess. In doing so the husFcγRIIB content after reconstitution wasadjusted to a nominal content of 60-180 mg/mL. The suitability of thevarious formulations for lyophilisation was evaluated based on thereconstitution time and the particulate contamination afterreconstitution. Several formulations were identified that could bereconstituted in less than 2 min and neither showed an increasedturbidity nor the formation of particulate in the visible andsub-visible range. Based on the above described lyophilisation screen itwas shown that ideal formulations contained low amounts of husFcγRIIB(e.g. 15-60 mg/mL) prior to lyophilisation and were reconstituted withlow volumes of water for injection, thereby increasing the finaldetergent concentration. The particulate load of selected formulationsas determined by fluorescence microscopy is shown in FIG. 10. Allformulations contained 5 mM citrate pH 6.7 and the indicated amount ofsalt, sugar and detergent. The formulations were lyophilized andreconstituted to the indicated husFcγRIIB content.

What is claimed is:
 1. A formulation comprising a soluble Fc

receptor (Fc

R) in a crystalline or microcrystalline form, 5 mM to 200 mM NaCl, 2.0%to 8% sugar, and a buffer solution comprising: (a) citrate at aconcentration of 1 to 50 mM, wherein the pH of the formulation is from5.2 to 5.9, or (b) histidine at a concentration of 1 to 50 mM, whereinthe pH of the formulation is from 6.0 to 7.5.
 2. The formulation ofclaim 1, wherein the receptor comprises an FcγRII receptor.
 3. Theformulation according to claim 2, wherein the receptor comprisesFcγRIIB.
 4. The formulation of claim 1, wherein said formulation furthercomprises a detergent.
 5. The formulation of claim 1, wherein saidformulation comprises a citrate buffered solution.
 6. The formulation ofclaim 1, wherein said formulation comprises a histidine bufferedsolution.
 7. The formulation of claim 1, wherein the Fc

receptor is present in a crystalline form.
 8. The formulation of claim1, wherein the Fc

receptor is present in a microcrystalline form.
 9. A method for treatingan autoimmune disease, comprising administering to a patient acomposition comprising the formulation of claim
 1. 10. The method ofclaim 9, wherein said administration comprises subcutaneousadministration.
 11. The method of claim 9, wherein said buffer solutioncomprises a histidine buffered solution.
 12. The method of claim 9,wherein said buffer solution comprises a citrate buffered solution. 13.The method of claim 10, wherein said subcutaneous administrationcomprises administration of a volume of 1.5 mL or less per application.